1. Field of the Invention
The present invention relates to a Bacillus bacterium which is useful for the production of purine-derived substances, including purine nucleotides such as 5′-inosinic acid and 5′-guanylic acid, and purine nucleosides such as inosine and guanosine. These nucleosides are important as starting materials for making purine nucleotides. Purine-derived substances are useful as seasonings, pharmaceuticals, and raw materials thereof.
2. Brief Description of the Related Art
Methods for producing inosine and guanosine by fermentation using adenine-auxotrophic mutants of Bacillus bacteria and derivatives thereof which are imparted with resistance to various drugs such as purine analogs (JP38-23099B, JP54-17033B, JP55-2956B, JP55-45199B, JP57-14160B, JP57-41915B, JP59-42895A, and US2004-0166575 A) have been previously described. Also, methods using mutants of Brevibacterium bacteria (JP51-5075B, JP58-17592B and Agric. Biol. Chem., 1978, 42, 399-405) have been described.
Such mutants are typically obtained by treating the cells with UV irradiation or N-methyl-N′-nitro-N-nitrosoguanidine, and selecting a target mutant in a suitable selective medium.
Strains which produce purine-derived substances have also been bred using genetic engineering techniques in Bacillus bacteria (JP58-158197A, JP58-175493A, JP59-28470A, JP60-156388A, JP1-27477A, JP1-174385A, JP3-58787A, JP3-164185A, JP5-84067A, and JP5-192164A), Brevibacterium bacteria (JP63-248394A), and Escherichia bacteria (WO99/03988). Specifically, a method of efficiently producing nucleic acid-derived substances such as hypoxanthine, uracil, guanine, and adenine with a Bacillus bacterium in which a gene (purR gene) encoding the purine operon repressor is disrupted is disclosed in U.S. Pat. No. 6,284,495.
In Bacillus subtilis, the purine operon repressor is known to regulate the expression of the purA. This gene is involved in AMP biosynthesis (Proc. Natl. Acad. Sci, USA, 1995, 92, 7455-7459). This repressor also regulates the expression of the glyA gene, which is involved formyltetrahydrofolate biosynthesis (J. Bacteriol., 2001, 183, 6175-6183), and the pbuG gene which encodes a hypoxanthine/guanine transporter (J. Bacteriol., 2003, 185, 5200-5209.), in addition to the purine operon gene.
A method for breeding a strain which efficiently produces inosine by disrupting the succinyl-AMP-synthase gene (purA) to impart adenine-auxotrophy and disrupting the purine nucleoside phosphorylase gene (deoD) to inhibit the decomposition of inosine into hypoxanthine in addition to disrupting the purR gene is described in US2004-0166575 A.
Meanwhile, in the oxidative pentosephosphate pathway, glucose is phosphorylated by glucose kinase to generate glucose-6-phosphate, which is oxidatively converted to ribose-5-phosphate. However, the relationship between this pathway and the biosynthetic pathway of purine-derived substances is not well understood; and therefore, it was not expected that a bacterium which produces purine-derived substances could be obtained by modifying the oxidative pentosephosphate pathway of the bacterium.